Abstract: Heterotrimeric G proteins are essential for transduction of extracellular signals into intracellular responses and therefore critical for the survival of multi-cellular organisms. Most neurotransmitters and hormones bind to cell surface receptors and activate G proteins that elicit cellular responses. As mediators between cell surface receptors and intracellular effectors, G proteins are well positioned to serve as targets of post-translational regulation. Emerging evidence indicates that G proteins are regulated by monoubiquitination, which has a well established role in membrane-protein internalization, trafficking and degradation. A ubiquitin ligase (Rsp5, ortholog of human Nedd4) has been discovered to be necessary and sufficient for monoubiquitination of the G-alpha subunit (Gpa1, ortholog of human G-alpha-i) in vivo and in vitro. The immediate objective of this proposal is to determine how Rsp5-catalyzed G protein monoubiquitination is regulated and how it affects fundamental properties of G protein activation and signaling. The long-term career goal is to become a principal investigator in the G protein research field with a focus on understanding the mechanisms of G protein regulation by post-translational modifications and their involvement in G protein signal transduction. In Aim 1 (K99 phase), the regulation of G-alpha monoubiquitination by heterotrimeric G protein assembly will be investigated. The hypothesis is that Gpa1 monoubiquitination and heterotrimeric complex assembly are mutually exclusive. This hypothesis predicts that G-beta/gamma binding inhibits Gpa1 monoubiquitination by Rsp5, and reciprocally, that Gpa1 monoubiquitination inhibits its re-association with G- beta/gamma. In Aim 2 (K99 and R00 phases), the role of phosphorylation on G-alpha monoubiquitination will be determined. Gpa1 is phosphorylated by multiple kinases at multiple positions surrounding the site of ubiquitination. The hypothesis is that phosphorylation is required for Gpa1 monoubiquitination. In Aim 3 (R00 phase), the regulation of G-alpha by de-ubiquitination will be investigated. De-ubiquitination of internalized proteins and recycling back to the plasma membrane has been shown to re-sensitize G protein signaling pathways. The ubiquitin protease Ubp12 has been implicated as a specific Gpa1 de-ubiquitinase. The hypothesis is that Ubp12 de-ubiquitinates Gpa1 and promotes its recycling. During the K99 phase of the award, significant training will be acquired in applied biophysical protein analysis (including structural, functional, and protein binding assays) through the Center for Structural Biology at the University of North Carolina at Chapel Hill (UNC). Through coursework, additional training will be acquired in fundamental principles of pharmacology. The research will be conducted in the departments of Biochemistry and Pharmacology at UNC, which has an exceptionally strong research infrastructure including world renowned G protein research faculty, outstanding research core facilities and superior educational opportunities. The ability to study G protein ubiquitination in vitro as well as in vivo is a unique advantage of the yeast experimental system. Not only does this allow in-depth research into the regulation mechanisms underlying G protein ubiquitination, but also into the effects of ubiquitination on the fundamental properties of G protein structure, function and protein interactions. Importantly, many strong parallels exist between the yeast system being studied here and humans. Therefore, the results if these studies will have a broad impact on human G protein research.